Confocal microscopy is a well-established technology with sub-micrometer lateral (perpendicular to the optical axis) and micrometer longitudinal (parallel to the optical axis) resolution. In a typical biomedical setting, this provides optical images of sections of tissue for qualitative and quantitative cellular morphology, pathology, and chemical analysis. The contrast and resolution of these images allows them to be compared to the gold standard of histopathological preparation and viewing of sectioned and stained tissue.
The use of Nomarski techniques applied to confocal microscopy, and especially laser scanning confocal microscopy, is known to enhance the contrast of objects with phase variations or surface profile variations. Such differential interference contrast (“DIC”) microscopes split a uniform, linearly-polarized or circularly-polarized illuminated pupil such that two point spread functions form at the focus of the objective. This is accomplished using a birefringent prism, such as a Nomarski or Wollaston prism, placed at the pupil (or conjugate location to the pupil) of the microscope. The prism shears the input beam, assumed to have polarization components both parallel and perpendicular to the Nomarski axis, into two beams and the orthogonally linearly-polarized pupils of the objective are focused to form two telecentric (in the object space) polarized spots. Upon reflection from the object, the sheared linearly-polarized beams are collected by the objective and re-combined at the pupil. Passing the recombined beam through a polarizing element provides an interference image, which is based on the phase profile of the scanned sample.
Such DIC configurations were previously improved by circularly-polarizing the sheared beams in order to further enhance the resulting image by reducing interference from turbidity above and below the section being imaged. See, for example, U.S. Pat. No. 6,577,394 to Zavislan, titled “Imaging System Using Polarization Effects to Enhance Image Quality.” FIG. 1 of the Zavislan patent depicts a prior art configuration of the polarization optics and objective of a microscope using a birefringement prism to shear a linearly-polarized beam into two linearly-polarized beams (having polarization orthogonal to each other) and a quarter wave plate retarder to circularly polarize the beams (opposite-handed polarization states).
By illuminating the sample with sheared beams having generally circular polarization in opposite senses (left and right handed circular polarization), images obtained using light returned from the image plane (i.e., a section within the sample), which may be altered by the sample's circular dichroism, retardation, etc., have reduced image distortion, such as that caused by scattering sites adjacent to the image plane or section.
Although prior techniques have improved the quality of images produced using microscopy, including confocal microscopy, there is a need for improved image quality by further reduction of noise caused by, for example, the turbidity of the medium.